Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder caused by a progressive loss of motor neurons (MNs), which results in death approximately 3-5 years after onset8,22. There is no cure for this devastating disease, and currently, there is only one FDA-approved drug, Riluzole, which prolongs life expectancy by a mere 2-4 months. microRNAs (miRNAs) are a promising new avenue for research that has not been adequately explored in neurodegeneration. These short, single-stranded non-coding RNAs regulate the expression of their mRNA targets through partial base-pairing facilitated by an Argonaute-containing protein complex9,10. miRNAs are often dysregulated in disease and have proven to be robust targets for therapeutic intervention11,13. Recently published results from my lab demonstrate that inhibiting miR-155 prolongs survival in ALS model mice by 38%14. However, a major limitation to developing effective miRNA- based therapeutics for ALS is a lack of understanding (MN) biology. Defining in vivo MN-specific miRNA expression patterns will help elucidate their selective vulnerability in ALS; however, characterizing MN-specific miRNAs requires miRNA expression in the other central nervous system (CNS) cell-types to be known. Using a novel affinity based approach, miRNAs associated with tagged Argonaute2 (Ago2) that is expressed only under cell-type specific promoters can be isolated17. I will apply this tool to define cell-type specific miRNA expression in the CNS of healthy mice to discriminate MN-specific miRNAs. I will also use this method to probe dysregulation of MN-specific miRNAs in ALS-model SOD1G93A mice. Furthermore, I have applied this tool to identify a potentially novel determinant of MN susceptibility in ALS. In Aim 1, I will use mice that express lox-stop-lox-GFP-myc-Ago2 crossed with mice that express Cre recombinase under cell-type specific promoters to define miRNA expression in all neurons, motor neurons, microglia and astrocytes. I will probe dysregulation of newly identified neuron- and MN-specific miRNAs in ALS by crossing the double transgenic mice into ALS model mice and analyze temporal miRNA changes in MNs throughout disease. In Aim 2, I will investigate the expression of a candidate miRNA highly upregulated in selectively vulnerable as compared to MNs resistant to ALS. I will determine the effect of ablating this miRNA on ALS disease progression by crossing previously generated knockout mice into the ALS mouse model. If successful, the proposed experiments will define MN-specific miRNAs, the dysregulation of which may inform MN disease mechanisms and serve as novel ALS biomarkers. In addition, these studies will elucidate the selective expression of a candidate miRNA in affected MNs and its relation to MN susceptibility in ALS.